Identifying Specific Bacteria -- How does it work?
MacConkey Agar
MacConkey Agar (MAC) is a selective and differential medium designed to isolate and differentiate enterics based on
their ability to ferment lactose. Bile salts and crystal violet inhibit the growth of Gram positive organisms. Lactose
provides a source of fermentable carbohydrate, allowing for differentiation. Neutral red is a pH indicator that turns red
at a pH below 6.8 and is colorless at any pH greater than 6.8.
Organisms that ferment lactose and thereby produce an acidic environment will appear pink because of the neutral red
turning red. Bile salts may also precipitate out of the media surrounding the growth of fermenters because of the
change in pH. Non-fermenters will produce normally-colored or colorless colonies.
SIM Test
SIM medium is a combination differential medium that tests three different parameters, which are represented by the
three letters in the name:
Sulfur Reduction
Indole Production
Motility
SIM medium contains nutrients, iron, and sodium thiosulfate. If an organism can reduce sulfur to hydrogen sulfide, the
hydrogen sulfide will combine with the iron to form ferric sulfide, which is a black precipitate. If there is any blackening
of the medium, it indicates the reduction of sulfur and is a positive result.
One of the nutrients in the SIM medium is peptone, which contains amino acids, including tryptophan. Some bacteria
possess the ability to produce the enzyme tryptophanase, which hydrolyzes tryptophan. The end products of this
hydrolyzation are indole, pyruvic acid, and ammonia, by way of deamination. The Kovac’s reagent that you add to the
SIM medium to test for indole contains hydrochloric acid, p-dimethylaminobenzaldehyde (DMABA), and n-amyl alcohol.
DMABA reacts with indole to produce a red quinoidal compound. If the reagent turns red, the indole test is positive.
Simmons Citrate Test
Simmons citrate agar tests the ability of organisms to utilize citrate as a carbon source. Simmons citrate agar contains
sodium citrate as the sole source of carbon, ammonium dihydrogen phosphate as the sole source of nitrogen, other
nutrients, and the pH indicator bromthymol blue.
Organisms which can utilize citrate as their sole carbon source use the enzyme citrase or citrate-permease to transport
the citrate into the cell. These organisms also convert the ammonium dihydrogen phosphate to ammonia and
ammonium hydroxide, which creates an alkaline environment in the medium. At pH 7.5 or above, bromthymol blue
turns royal blue. At a neutral pH, bromthymol blue is green, as evidenced by the uninoculated media. If the medium
turns blue, the organism is citrate positive. If there is no color change, the organism is citrate negative. Some citrate
negative organisms may grow weakly on the surface of the slant, but they will not produce a color change.
Christensen Urea Test
Urea Agar was devised with peptone, dextrose, and a reduced buffer to promote more rapid growth of many of the
Enterobacteriaceae and permit a reduction in incubation time. The complete Urea Agar contains 15.0 g/L of agar in
addition to the ingredients in the base medium including: Pancreatic Digest of Gelatin, Sodium Chloride, Potassium
Phosphate, Urea, and Phenol Red.
When organisms utilize urea, ammonia is formed during incubation which makes the reaction of these media alkaline,
producing a red-pink (or red-violet) color. Consequently, urease production may be detected by the change in the
phenol red indicator. The color may penetrate into the agar (into the butt of the test tube); the extent of the color
indicates the rate of urea hydrolysis. A negative reaction will have no color change and the agar medium will remain
pale yellow to buff.